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Probiotics for Frogs

Healthy bacteria may be the cure to amphibian decline worldwide

Katie L. Burke

2013-05SciObsBurkeFA.jpgClick to Enlarge ImageMostly out of sight, and perhaps out of mind also, the world has been losing its amphibians to a disease called chytridiomycosis, caused by the chytrid fungus (Batrachochytrium dendrobatidis). Due to this epidemic, at least 200 of the world’s 6,700 amphibian species have gone extinct. A third of the species left are considered threatened by the International Union for Conservation of Nature. Even more than that—40 percent—are declining. The scope of the problem is worse than the plague was for humans in the Middle Ages; during the Black Death, almost a quarter of the human population is estimated to have been lost. So imagine how exciting it would be to figure out a possible mitigation strategy.

Imagine that you are a biologist in the Middle Ages—even though that occupation did not really exist back then. Amid the adversity and mortality of the Black Death epidemic, you figure out that what everyone needs is a particular culture of bacteria, and if they have it, odds are they will live. Envision having the capacity to curb that loss of life—and having to convince people that germs are the cure. Reid Harris of James Madison University has a similar problem, except he studies the contemporary amphibian pandemic, not the medieval human one.

Three years ago, Harris, along with Vance Vredenburg of San Francisco State University, had a windfall discovery: They found in a laboratory experiment that the presence of a certain bacterium called Janthinobacterium lividum reliably predicted whether endangered frogs in the Sierra Nevada Mountains would survive the chytrid fungus epidemic. Field trials inoculating the frogs with the bacterium also proved successful. J. lividum produces a substance called violacein that inhibits chytrid fungal growth. This research has served as a springboard for studying potential probiotic treatments for chytrid fungus worldwide. J. lividum is very widespread—it is found on the skin of amphibians in North America, Central America, South America, Europe and perhaps other continents. The presence and effect of J. lividum raise the question of what other probiotics are out there in the microbiomes on amphibian skin around the world.

In a recent review paper in Ecology Letters, Harris—with his master’s degree students Molly Bletz and Andy Loudon as well as other collaborators in the fields of genomics and organic chemistry—laid out a plan for developing disease mitigation protocols. Several problems immediately reared their heads: How do you choose what to save first from more than 6,000 species? And out of the thousands of bacterial species on an amphibian’s skin, where do you start looking for a probiotic? Additionally, how do you make sure that introducing a bacterial culture is not going to harm the ecosystem overall? Furthermore, how do you convince people that bacteria are good?

“So many frogs, so little time,” Harris says. He could have said the same about the bacteria. “We may be able to treat amphibians’ breeding ponds with a probiotic, and then it would be picked up by a variety of species,” Harris continued. “The species-specific treatment approach is going to be important for very charismatic or endangered species, but it would be nice to develop a more community-based approach as well.” To do so, the authors assert, the probiotic used must be from the environment where the treatment will happen and should not have side effects of concern. “Using probiotics that are naturally found on amphibians wherever the application is planned to be will reduce the risk of causing any ecosystem problems compared to something that is nonnative,” Bletz said. Her preliminary work has found no effects of J. lividum treatment in ponds on leaf decomposition, zooplankton community composition or periphyton production (a measure of growth in algae, cyanobacteria and bacteria). She is currently testing effects on the ponds’ bacterial communities. The antifungal properties of J. lividum may only be “turned on” when in the context of the amphibian’s skin, which would mean that the bacteria in the water would not have major effects on other organisms. “Frogs produce antimicrobial peptides,” Harris explained. “In an experiment with antimicrobial peptides from Rana muscosa, the mountain yellow-legged frog, and a potential probiotic called Pseudomonas, some of the combinations were synergistic, so you needed really low amounts of both to get a nice inhibitory effect on chytrid. There’s potential for cooperation between the defensive molecules produced by the frog and the defensive metabolites produced by the bacteria.”

2013-05SciObsBurkeFB.jpgClick to Enlarge ImageTo tackle the huge numbers of bacteria, these biologists have technology on their side. The group is using the latest genomics technology, called Next Generation sequencing, which sequences more DNA faster and more accurately than previous techniques. “With Next Generation sequencing, you get massive amounts of data. For instance, in recent research with Valerie McKenzie and Rob Knight of the University of Colorado, our dataset had over 23 million sequences, and that was for just 65 salamanders and their respective environments,” Loudon notes. It’s not just the sheer number that is astounding but also that the microbiome is dynamic: “If you define stability as maintaining the same microbial species and the same abundances over time, the trend with microbiomes is like looking at a moving picture. They do not stay the same over time,” Loudon explains. By sequencing the microbiome from amphibians that are surviving with chytrid fungus present, they hope to find potential probiotics that can be screened in inhibition assays (checking for a bacterial colony’s ability to inhibit growth of the fungus), then lab experiments and then field trials. As Harris commented, “Our work has paralleled the Human Microbiome Project, which is also identifying natural or notable microbiota, how variable the microbiome is, how protective it is and so forth. Of course, they are relatively well funded and are working on one host species, humans. Our work is a massive community ecology project, even to get this worked out on one endangered host frog species.”

Harris and Bletz are focusing efforts on an amphibian hotspot that is currently free of chytrid fungus, or as Harris put it “a ticking time bomb”—Madagascar. “Given the way chytrid fungus has spread around the world, it’s probably only a matter of time before it arrives in Madagascar,” Harris remarked. “Ché Weldon of North-West University has done some testing on a handful of Malagasy species, and they are susceptible. It is imperative to get over there and develop a bank of potentially useful probiotics now, so we would have some mechanism of preventing the epidemic spread.” By studying Madagascar before and during infection with chytrid, they will collect valuable knowledge about the disease’s dynamics to help predict and prevent infections in the future. The potential to save a lot of frogs while focusing on a defined area makes the project a high priority. “There are 400 species of amphibian in Madagascar; some of those have yet to be described. Ninety-nine percent of these amphibians are endemic. Maybe the bacteria are endemic as well,” says Bletz. She and Harris are working on putting together a team of collaborators, developing infrastructure for research in Madagascar and raising funds for it. They want to get local communities involved: “A lot of people still think bacteria are bad, and that’s not the case. A hurdle to jump over will be informing communities about what we’re doing,” Bletz adds.

Harris agrees that there would not be one magic silver bullet to end all chytrid fungus problems. But he notes: “I’m cautiously optimistic. If it’s worked in one species, the mountain yellow-legged frog, it gives a lot of encouragement that we can figure out how to make it work in other species.” It’s a good thing it’s not the Middle Ages. Sterile techniques and the scientific method, not to mention Next Generation sequencing, may save the large majority of a class of animals from the brink of population decline and even extinction.

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